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均一颗粒理化性质与佐剂效应关系的研究
Alternative TitleStudy on relationship between physicochemical properties and adjuvanticity of uniform-sized particles
刘媛
Subtype博士
Thesis Advisor马光辉
2014-04
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword颗粒型佐剂   亲疏水性   表面基团   粒径效应   免疫应答
Abstract随着近年来生物技术和基因工程的发展,众多新型疫苗被开发出来,这些疫苗安全性越来越高,但同时伴随着免疫原性的降低,因此需要向疫苗中添加佐剂这种非特异性免疫增强剂,才能构成安全且高效的疫苗。而颗粒型佐剂正是目前在研的佐剂中最有前景的佐剂之一。为了合理设计颗粒型佐剂,首要任务是针对佐剂的特定需求,考察清楚颗粒的理化性质对其佐剂效果的影响。因此,本论文基于佐剂的作用机理,采用模型抗原,通过快速膜乳化技术制备粒径均一可控的颗粒,考察了不同理化性质对颗粒与免疫系统中关键成分相互作用的影响,从机制上阐述了颗粒理化性质与其佐剂效应的关系,为今后新型颗粒型佐剂的设计和构建奠定了基础。 具体研究内容包括以下几个方面: (1)采用聚乳酸类材料制备粒径均一的颗粒,从非特异性免疫系统中重要组成部分树突状细胞DC入手,研究了颗粒表面疏水性对其佐剂效果的影响。采用快速膜乳化技术成功制备出三种具有不同亲疏水性的颗粒,它们粒径均一并拥有相似的其他物化性质。通过考察细胞摄取、细胞活化、细胞迁移以及免疫动物后的免疫应答水平,发现随着颗粒表面疏水性的增强,颗粒的佐剂效果也随之增强。通过原子力显微镜对细胞与颗粒间相互作用力的原位分析,揭示了作用机制,发现颗粒疏水性的增强将增大颗粒与细胞间的相互作用力,进而推动细胞的摄取行为,最终增强免疫应答 (2)采用天然多糖类材料制备粒径均一的颗粒,从非特异性免疫系统中重要组成部分补体系统入手,研究了颗粒表面基团对其佐剂效果的影响。结合快速膜乳化技术和层层自组装技术,成功制备出两种具有不同表面基团的颗粒(CS-NH2和CS-CL),其中,CS-NH2颗粒表面富含伯胺,CS-CL颗粒表面富含叔胺,它们粒径均一并拥有相似的其他物化性质。通过补体活化实验和动物体内免疫学评价,发现与表面富含叔胺的CS-CL颗粒相比,表面富含伯胺的CS-NH2颗粒能够更加有效地活化补体系统,并通过补体活化途径进一步增强其佐剂效果。 (3)基于上述CS-NH2颗粒能够通过活化补体进而增强机体免疫应答的基础上,为了进一步增强颗粒的佐剂效果,向颗粒中添加了TLR7/8激动剂咪喹莫特IMQ,针对激活非特异性免疫系统的作用机理,初步探索了以该颗粒作为疫苗佐剂的可行性。通过细胞水平实验发现,通过加入TLR激动剂可显著增强DC细胞和巨噬细胞的活化水平。以这种具有双重刺激功能的颗粒作为疫苗佐剂,具有值得期待的前景。 (4)采用快速膜乳化技术制备了粒径均一的疫苗,与高速机械搅拌法制备的疫苗对比,从抗原储库效应的作用机理入手,考察不同粒径均一性在抗原释放、免疫细胞募集以及诱导免疫应答水平等方面的影响,发现采用快速膜乳化技术制备的乳剂具有极低的突释率,且在后续的释放中能够维持较为平稳和快速的释放,同时,快速膜乳化技术制备的疫苗不论是HI抗体还是抗原特异性IgG抗体均显著高于高速机械搅拌法制备的疫苗,同时通过进行重复性实验,验证了实验结论的普适性,展示了快速膜乳化技术应用于油乳剂疫苗制备的优越性。 本论文的研究结果表明颗粒的表面亲疏水性、表面伯胺和叔胺基团以及粒径均一性均对其佐剂效果有显著影响,通过调控以上性质,可以更合理地设计和构建出高效的颗粒型疫苗佐剂。
Other AbstractWith the advent of genetic engineering and biotechnology, numerous safe vaccines have been developed using recombinant proteins, protein subunits, or synthetic oligopeptides. However, because of their weak immunogenicity, vaccines also require adjuvant to enhance immune responses. Over the last two decades, an increasing number of studies showed that microparticles (MPs) and nanoparticles (NPs) can function as effective adjuvants. To rationally design particulate adjuvants, the primary task is exploring the relationship between physical and chemical properties of particles and their adjuvant effects. We systemically investigated the effects of particles surface hydrophobicity, surface groups, and size uniformity on their adjuvant effects using model antigen. The results explained the relationship between physicochemical properties and adjuvanticity of uniform-sized particles based on the action mechanism, laid the foundation for the design and construction of the new particulate adjuvant. In detail, this thesis mainly included the following issues: (1) To study the correlation between particle hydrophobicity and their adjuvanticity in-depth, we prepared three kinds of particles by premix membrane emulsification, which were similar in size and morphology but differed in surface hydrophobicity. Starting with dendritic cells (DCs), an important part in nonspecific immune system, we systematically evaluated their ability to induce immune responses in vitro and in vivo. Increased surface hydrophobicity on PLA-based particles greatly promoted antigen internalization into dendritic cells (DCs) as well as MHC II and CD86 expression on DCs in vitro. Similarly, in vivo studies showed that increased particle surface hydrophobicity significantly elevated cytokine secretion levels by splenocytes harvested from vaccinated mice. Adhesion force measurements confirmed that increased surface hydrophobicity enhanced the physical interaction between particles and cell membranes, a condition favorable for promoting particle internalization into cells. (2) To study the correlation between particle surface chemical groups and their adjuvanticity, we prepared two kinds of particles by premix membrane emulsification, which were similar in size and morphology but differed in surface chemical groups. CS-NH2 particle surfaces are rich in primary amine, while CS-CL particle surfaces are rich in tertiary amine. Starting with complement system, an important part in nonspecific immune system, we evaluated their ability to active complement system and the ability to induce immune responses. We found that CS-NH2 particles with a high surface density of primary amine were better able to induce complement system activation. In vaccination studies, we found that particles with abundant primary amine significantly enhanced antigen-specific immune response. Overall, these results indicated that CS-NH2 particles with a high surface density of primary amine contributed to complement activation and immune responses. These results provide further design principles for studies of complement-activating particles as potential vaccine platforms. (3) Having revealed CS-NH2 particles with a high surface density of primary amine contributed to complement activation and immune responses, we continued to add the TLR7/8 agonists imiquimod (IMQ) into CS-NH2 particles, to further enhance the adjuvant effect of particles. In vitro studies, we found there is a significant enhancement in DCs and macrophages activation by adding the TLR agonist. Therefore, it had extensive application prospect by using this particle of dual stimulating functions as adjuvant. (4) To study the correlation between particle size uniformity and their adjuvanticity, we prepared uniform-sized emulsion by premixed membrane emulsification technique and non-uniform emulsions by mechanical stirring. Starting with antigen depot effect, the one mechanism of adjuvant action, we investigated the effect of emulsion
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15543
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
刘媛. 均一颗粒理化性质与佐剂效应关系的研究[D]. 中国科学院研究生院,2014.
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